The term “collagen” is defined as protein or glycoprotein, which has at least partially a helical structure (collagen helix) This is a triple helix composed of three polypeptide chains, wherein each polypeptide chain has a molecular weight of about 100,000 in which a glycine residue appears every three residues and proline and hydroxyproline residues appear as other amino acid residues at high frequency. Collagen can be extracted in a large amount from the tissues of an invertebrate or a vertebrate animal, in particular from the skin thereof. It has been reported that collagen molecules can be divided into 19 different types depending on their different structures and the collagen molecules classified in the same type may include several different molecular species.
Particularly, types I, II, III and IV of collagen have been mainly used as sources of biomaterials. The type I can be found in most of the connective tissues and is the most abundant collagen type in a living organism, particularly in the tendon, corium and bone. Industrially, in many cases, collagen can be extracted from those portions. The type II is a collagen that forms a cartilage. The type III is found in a small quantity in the same portions as those of the type I. The type IV is a collagen that forms a basal membrane. The types I, II and III are present as collagen fibers in a living organism, and have a main function of maintaining the strength of a tissue or of an organ. The type IV, which does not have ability for fibrogenesis, forms a net-like assembly constructed of four molecules and may take part in cell differentiation in a basal membrane. In the present specification, hereinafter, the term “collagen” refers to one of the types I, II and III or a mixture of two or more of them.
A collagen fiber is a self-assembly of the collagen molecules and has a specific fiber structure in which the collagen molecules are packed in series and parallel. Industrially, using acid, alkali or a proteolytic enzyme, solubilized collagen can be prepared from collagen fibers in tissues.
The solubilized collagen may be divided into assemblies of not more than several collagen molecules and then dissolved in water or an aqueous salt solution to prepare a uniform transparent solution. It is known that collagen molecules, which are once solubilized, will regenerate collagen fibrils in vitro under certain conditions. This phenomenon is called fibril formation (fibril formation or fibrillation) and the detailed characteristic features thereof are described in Biochemical Journal 316, p. 1-11 (1996).
When collagen is heated, the triple helix structure of collagen comes loose and then each polypeptide chain provides a thermal denatured product in a random coil form. The temperature at which such a structural change occurs is called “denaturation temperature”, while the thermal denatured product is called “gelatin”. It is known that gelatin has higher water solubility than that of collagen and high sensitivity to in vivo protease. Depending on the solvent conditions, gelatin is known to partially recover a collagen helical structure. Even though gelatin has lost its ability of forming a collagen fiber, it is known that it may recover its ability of forming a collagen fiber by partially recovering its collagen helical structure.
The denaturation temperature of collagen is the lowest in a solution state. In addition, collagen is generally obtained from a biological source and the denaturation temperature of collagen obtained from a living organism is allegedly closely associated with environmental temperatures surrounding the organism. Denaturing temperature of collagen of mammals in an aqueous solution is about 38° C. The denaturation temperature of collagen of fishes is generally lower than that of the collagen of mammals and in particular fishes living in cold currents, such as salmon, may each have a denaturation temperature of lower than 20° C.
Collagen has superior moisture retention and is cheaper than other biogenetic humectants such as hyaluronic acid because of its higher yield. Therefore, it is effectively used as a raw material in cosmetics. In addition, collagen has many excellent properties such as facilitating adhesion and growth of cells, low antigenicity, high bioaffinity and biodegradability, so that it has been used in various kinds of applications, such as materials for experiments on cells and medical materials. When collagen is used in any of those purposes, it may be used in any of various forms such as an aqueous solution, floc, film, sponge and gel depending on the applications. In particular, collagen gel is effectively used for cell carriers, medical materials, and soon. In recent years, extensive studies have been conducted on collagen gel as an important material in regenerative medicine. Processes of producing collagen gel can be divided into three types as follows.
1. A process in which a cross-linking agent is introduced into a collagen solution to gelatinize the solution.
2. A process in which a collagen solution is irradiated with luminous rays inducing cross-link to gelatinize the solution.
3. A process in which a neutral buffer is added to a collagen solution to induce fibril formation of collagen to obtain a gel constructed of a collagen fibril network.
Regarding the above-mentioned process 1, for example, there are disclosed a collagen gel formed product for ophthalmology obtained by mixing a chemical cross-linking agent in a collagen solution to gelatinize the solution (JP-A-11-197234) and a gel for tissue-regenerating matrix in which a mixture solution of glycosaminoglycan and collagen cross-linked with aqueous carbodiimide (JP-A-2002-80501). Regarding the above-mentioned process 2, for example, there is a report that a collagen solution is gelatinized when it is sufficiently substituted with nitrogen and then irradiated with ultraviolet rays (Biochimica Biophysica Acta 229, p. 672 to 680 (1971)). Regarding the above-mentioned process3, for example, a gel constructed of a collagen fiber network is reported which is obtained by mixing an aqueous solution of shark-originated collagen with a neutral buffer to induce fibrosis of collagen (Journal of Agricultural Food Chemistry 48, P. 2028 to 2032 (2000)).
The collagen gels made by the above three gelation processes are lacking in sufficient thermal stability. In a certain application, collagen may be denatured to cause softening or dissolving of the gel to thereby become unusable. In addition, the strength of the gel is insufficient, so that in a certain application the gel may be constricted and degraded to thereby become unusable.
Therefore, in recent years, in view of improving the strength of a collagen gel, there has been disclosed technology to bring a collagen gel prepared by fibril formation of an acidic collagen solution into contact with a protein-linking agent (JP-A-8-283667). However, in this technology, the cross-linking takes place only on the surface of the collagen fiber fibril and the cross-linking agent does not reach the central part of the gel, resulting in poor improvement in strength. Consequently, there is a problem that the technology does not attain a substantial improvement with respect to the thermal stability of gel.
Any one of the cross-linking agents known in the art has not succeeded in solving an essential problem in that it does not act on the inside of a helix of a collagen molecule but act on between the collagen molecules and another problem that the cross-linking agent hardly exerts its effect on the inside of a collagen gel. Therefore, an improvement in thermal stability has not been attained sufficiently. In other words, there has been no disclosure with respect to a technology that imparts higher thermal stability to a collagen gel than those of the collagen gels prepared by the above three gelation processes.
Furthermore, most of collagen as a raw material for a collagen material has been conventionally collected from the tissues of domestic animals, such as bovine hide. However, in recent years, the issue of BSE (bovine spongiformencephalopathy) has been elicited. It has been pointed out that a risk of infections of pathogens to humans lie latent in collagen products using raw materials from domestic animals including bovine hide. From the viewpoints of safety, quantity of natural resources, and so on, fish-origin collagen comes into the limelight as any of cosmetic and food materials. Thus, it becomes important to use fish-origin collagen obtained from fishes having a low denaturation temperature as a raw material of collagen.
However, though the fish-origin collagen has a low risk, due to its low denaturation temperature, its thermal stability as a material is insufficient in many cases. Therefore, comparing with collagen originated from a domestic animal, the fish-origin collagen may be disadvantageous as a raw material of a cell carrier or of a medical material.
The above-mentioned problems with respect to insufficient thermal stability or strength, and the like in the conventional process of producing collagen have restricted a wide variety of applications of general collagen gels originated from domestic animals to medical materials. Furthermore, for using the gels as medical materials, the gels are often requested to be stable at least at a body temperature of 37° C., so that the conventional process was insufficient for a stabilization approach for collagen gels derived from fishes.